Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Insulin-dependent type 1 diabetes is caused by lack of insulin, due to autoimmune-mediated destruction of pancreatic islet cells. People with type 1 diabetes need regular insulin injections to control their blood glucose level, a matter of life or death. Pancreas transplantation is currently the only curative therapy for type 1 diabetes, but it is hampered by the requirement for potentially toxic, life-long immunosuppressive drugs and by the dearth of human donors. These barriers could be overcome by discovery of growth factors to (re)generate cells.
The factors and mechanisms which regulate lineage differentiation of islet cells from multi-potent precursors are poorly understood. Broadly speaking, two classes of co-operative signals guide cellular proliferation, differentiation and apoptosis: soluble signals including hormones and growth factors and insoluble signals delivered by extracellular matrix (ECM) proteins (Lelievre et al., 1996). Classical hormones play important roles in regulating endocrine cell development. For example, pituitary luteinizing hormone (LH) controls lineage differentiation of Leydig cells in the testis and of granulosa cells in the ovary, while thyroid stimulating hormone (TSH) and adrenocortico trophic hormone (ACTH) maintain differentiation of thyroid follicular cells and adrenocortical cells, respectively. Islet cell development seems to be an exception, as no hormonal control has been demonstrated.
The mammalian pancreatic primordia evaginate from foregut endoderm in early fetal life. The adult pancreas consists of two distinct tissue types: endocrine tissue, the islets of Langerhans, which secretes hormones into the bloodstream, and exocrine tissue, which secretes digestive enzymes into the intestinal tract. The islets contain four main types of endocrine cells that synthesize insulin, glucagon, somatostatin and pancreatic polypeptide. These hormones, notably insulin, play critical roles in glucose metabolism and homeostatis. All four types of endocrine cells are believed to arise from common multi-potent precursors which express the PDX-1 (also called IPF-1, STF-1 and IDX-1) transcription factor and co-express several hormones and neuronal markers as they begin to differentiate (Slack, 1995). Several soluble extracellular factors have been implicated in pancreatic epithelial cell development, including members of the TGF-β1 superfamily of transforming growth factors. Transgenic mice expressing a dominant negative TGF-β receptor II controlled by the mouse metallothionein 1 promoter display increased proliferation and impaired differentiation of pancreatic acinar cells (Bottinger et al., 1997). Transgenic mice expressing a dominant negative activin receptor controlled by the human insulin promoter have hypoplasia of pancreatic islets (Yamaoka et al., 1998). Hebrok et al. (1998) found that Activin B is expressed in the notochord adjacent to the domain of foregut endoderm from which the pancreatic primordia derives. Activin B represses endodermal expression of sonic hedgehog, a prerequisite for expression of the homeodomain transcription factor, PDX-1, required for pancreatic development (Jonsson et al., 1994; Offield et al, 1996). The bone morphogenetics (BMPs), members of the TGF-β superfamily, have been shown to be important in development of kidney tubule, lung and other organ epithelia (Hogan, B. L, 1996; Weaver et al., 1999) and are expressed in the pancreas. BMP 7 was detected immunocytochemically in human fetal pancreas duct epithelium (Vukicevic et al., 1994) and by mRNA in situ hybridization in mouse pancreas epithelium between E12.5 and E14.5 (Lyons et al, 1995). The appropriate and timely expression of these factors contributes to the appropriate embryonic development of the pancreas.
Laminin-1 is a heterotrimeric cellular matrix glycoprotein (Mr=850,000) composed of (400 kDa), (210 kDa) and (200 kDa) disulfide-bonded chains (Ekblom, 1996). Laminin-1 has been shown to induce specifically β-casein gene expression in mammary epithelia (Streuli et al., 1995) and neuron generation from retinal neuroepithelial cells (Frade et al., 1996). The cross region of laminin-1 selectively promotes fetal lung epithelial cell proliferation, the outer globular region of the α1 and β1 chains mediates epithelial cell polarization, and the inner globular region of the β1 chain binds to heparin sulfate proteoglycan and stimulates lumen formation (Schuger et al., 1996). There are at least two types of laminin-1 receptor: the α6 integrins and the non-integrin α-dystroglycan (αDG) (Ekblom, 1996). Integrins are a well-characterized family of heterodimeric cell adhesion molecules composed of non-covalently bound (120-180 kDa) and (90-110 kDa) subunits. αDG is a 156 kDa extracellular peripheral membrane glycoprotein associating with a transmembrane glycoprotein, which binds laminin-1 with high affinity but does not bind nidogen, fibronectin or collagen IV (Ekblom, 1996). The present inventors have found by RT-PCR that mRNAs for α6 integrin and αDG are expressed in the developing mouse pancreas from at least 13.5 dpc and α6 integrin protein is detected by immunofluorescence from at least 15.5 dpc. Coincident with these laminin-1 studies, the present inventors also carried out a representational difference analysis (RDA) in which genes expressed in normal human pancreas were subtracted from genes expressed in pancreas from a child with diffuse islet cell hyperplasia (nesidioblastosis). One of the genes found to be differentially expressed in nesidioblastosis was bone morphogenetic protein (BMP) 7. BMPs were originally identified as proteins that induce bone and cartilage formation in ectopic extraskeletal sites in vivo (Wozney, 1989). In vitro studies have revealed that BMPs have multiple effects on various cells types. BMP-2 deficient mice have amnion/chorion malformation and defective cardiac development, and die between 7.5-9 dpc (Zhang and Bradley, 1996). BMP 4 deficient mice have defects in extra-embryonic and posterior/ventral mesoderm formation and die between 6.5-9.5 dpc (Winnier et al., 1995). BMP 7 deficient mice have defects in kidneys and eyes and die shortly after birth (Dudley et al., 1995; Luo et al., 1995).
In work leading up to the present invention, the inventors developed a low cell density, serum-free culture system for dissociated pancreatic cells from 13.5 day-postcoitum (dpc) mouse fetuses and investigated the effects of four major ECM proteins, collagens I and IV, fibronectin and laminin-1, on the differentiation of fetal pancreatic cells into islet cells (Jiang et al., 1999). Following four days of culture in complete HYBRIDOMA medium, the total cell number decreased to one-third of that plated, but the number of insulin-positive cells increased 10-fold. Both collagens I and IV inhibited (by over 50%) the survival of pancreatic cells compared to medium alone, whereas fibronectin had no effect. However, in the presence of soluble laminin-1, the number of cells increased linearly by 60-fold. Laminin-1 was also shown to be expressed in the epithelial basement membrane of the 13.5-17.5 day fetal pancreas (Jiang et al., 1999). These results provided the first evidence that laminin-1 plays an important role in promoting differentiation of pancreatic cells. The present inventors have further developed an in vivo culture system in which the interaction of laminin-1 with particular BMPs has a synergistic effect which result in an increased frequency in the formation of cystic epithelial colonies that contain insulin-producing cells. The subject inventors showed that they are able to regulate the development and formation of cystic epithelial colonies which contain cells that express insulin by modulating the quantity of particular BMPs and/or laminin-1. The present inventors further identified that TGF β1 and Activin A antagonize the activity of particular BMPs. Thus, the present inventors are now able to regulate the development of insulin-producing β-cells from pancreatic epithelial cells in vitro. The present invention further permits the development of protocols for treating diabetes as well as conditions such as β cell hyperplasia, nesidioblastosis and pancreatic cancer.